Surface Enhanced Raman spectroscopy (SERS) is a sensitive, instant and nondestructive sensing technique that has been widely used in analytical chemistry, environmental protection and food safety. However, the samples in real-world usually contain many different components, which makes the identification of analytes from mixed samples very difficult for SERS sensing. For their advanced features such as simple operation process, cost-effective and instant separation time, thin layer chromatography (TLC) technology was widely employed in synthetic chemistry, analytical chemistry, medicinal chemistry and food science as a powerful separation strategy. The target system was firstly separated by TLC, and the corresponding spots of analyte are visualized by iodine colorimetry or fluorescence, and then the analytes werequalitative and quantitative identified by mass spectrometry, FTIR, fluorescence spectroscopy and SERS. The in-tandem of SERS with thin layer chromatography (TLC-SERS) is very promising in effectively sensing analytes from mixture samples due to its high throughput and sensitivity and the separation ability. The complicated apparatuses are not necessary during the application process of TLC-SERS. This paper introduced the mechanism of SERS enhancement and the preparation of SERS substrates, and this review highlights recent progress of TLC-SERS in the view from the application, such as in monitoring pollutants in the environment, food safety, identification of herbal medicine and biomedicine. The application example of TLC-SERS method on instant inspection of harmful ingredients was then presented, which provides a reference for future instant inspection method construction and development of rapid detection instruments in the area of food safety, forensic characterization and environmental protection.